Compositions and methods for transplantation of colon microbiota

ABSTRACT

The present invention provides compositions that include an extract of human feces, and methods for using such compositions, including methods for replacing or supplementing or modifying a subject&#39;s colon microbiota, and methods for treating a disease, pathological condition, and/or iatrogenic condition of the colon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/594,087 filed May 12, 2017, which is a continuation of U.S.application Ser. No. 15/261,319 filed Sep. 9, 2016, now U.S. Pat. No.9,649,343 issued May 16, 2017, which is a continuation of U.S.application Ser. No. 14/003,411 filed Jan. 17, 2014, which is a 371 ofInternational Application No. PCT/US2012/028484 filed Mar. 9, 2012,which claims the benefit of U.S. Provisional Application No. 61/450,838filed Mar. 9, 2011, the entireties of each of which are hereinincorporated by reference. U.S. application Ser. No. 15/594,087 is alsoa continuation of U.S. application Ser. No. 15/173,134 filed Jun. 3,2016, which is a continuation of U.S. application Ser. No. 14/003,411filed Jan. 17, 2014, which is a 371 of International Application No.PCT/US2012/028484 filed Mar. 9, 2012, which claims the benefit of U.S.Provisional Application Ser. No. 61/450,838, filed Mar. 9, 2011, theentireties of each of which are herein incorporated by reference. Thisapplication is also a continuation of U.S. application Ser. No.15/594,319 filed May 12, 2017, which is a continuation of U.S.application Ser. No. 15/261,319 filed Sep. 9, 2016, now U.S. Pat. No.9,649,343 issued May 16, 2017, which is a continuation of U.S.application Ser. No. 14/003,411 filed Jan. 17, 2014, which is a 371 ofInternational Application No. PCT/US2012/028484 filed Mar. 9, 2012,which claims the benefit of U.S. Provisional Application No. 61/450,838filed Mar. 9, 2011, the entireties of each of which are hereinincorporated by reference. U.S. application Ser. No. 15/594,319 is alsoa continuation of U.S. application Ser. No. 15/173,134 filed Jun. 3,2016, which is a continuation of U.S. application Ser. No. 14/003,411filed Jan. 17, 2014, which is a 371 of International Application No.PCT/US2012/028484 filed Mar. 9, 2012, which claims the benefit of U.S.Provisional Application Ser. No. 61/450,838, filed Mar. 9, 2011, theentireties of each of which are herein incorporated by reference.

GOVERNMENT FUNDING

This invention was made with government support under AI091907 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

In 1978, Clostridium difficile was first recognized as a major cause ofdiarrhea and pseudomembranous colitis associated with the use ofantimicrobial agents. Since this time, infection by C. difficile hasbeen steadily growing in incidence, morbidity, and mortality acrossNorth America and Europe (Freeman et al. Clin Microbiol Rev 2010;23:529-49; Kelly and LaMont, N Engl J Med 2008; 359:1932-40). Analysisof the U.S. National Hospital Discharge Survey statistics between 1996and 2003 reveals a doubling in the prevalence of diagnosis of C.difficile infection (CDI), to 0.61/1,000, among inpatients (McDonald etal. Emerg Infect Dis 2006; 12:409-15). A 2008 survey of 12.5% of allU.S. acute care facilities indicated a CDI prevalence rate of13.1/1,000, which is at least an order of magnitude higher than thatfound previously (Jarvis et al. Am J Infect Control 2009; 37:263-70).While older patients have disproportionately greater rates of CDI thanyounger individuals, no age group is spared, and the incidence ofCDI-related hospitalizations has been rising even in the pediatricpopulation (Zilberberg et al. Emerg Infect Dis 2010; 16:604-9). Theincrease in incidence has been further compounded by an elevatedfrequency of the most severe forms of this disease, as evidenced byrising CDI-associated morbidity and case fatality (Ricciardi et al. ArchSurg 2007; 142:624-31; discussion 631, Zilberberg et al. Emerg InfectDis 2008; 14:929-31). This is, in part, related to the emergence of morevirulent C. difficile strains, such as PCR ribotype 027/North AmericanPulsed Field type 1 (NAP1), which is characterized by a greaterpotential for toxin production and antibiotic resistance than otherclinically-relevant strains (Rupnik et al. Nat Rev Microbiol 2009;7:526-36, Kuijper et al. Euro Surveill 2008; 13).

Recurrent CDI is one of the most difficult and increasingly commonchallenges associated with CDI (Surawicz, Gastroenterology 2009;136:1152-4). An initial incidence of CDI can be followed by a relapsewithin 30 days in about 20-30% of cases (Kelly and LaMont, N Engl J Med2008; 359:1932-40 (2008); Louie et al. N Engl J Med 2011; 364:422-31;Pepin et al. Clin Infect Dis 2006; 42:758-64), and the risk ofrecurrence doubles after two or more occurrences (McDonald et al. EmergInfect Dis 2006; 12:409-15). Older age, intercurrent antibiotic use fornon-C. difficile indications, renal insufficiency, immune deficiency,and antacid medications, are some of the known risk factors forrecurrent CDI (Surawicz, Gastroenterology 2009; 136:1152-4, Garey et al.J Hosp Infect 2008; 70:298-304). The presence of just three clinicalcriteria: age >65 years, severe disease, and continued use ofantibiotics after treating the initial CDI episode, are predictive of analmost 90% relapse rate (Hu et al. Gastroenterology 2009; 136:1206-14).CDI also commonly complicates management of inflammatory bowel disease(IBD), which has recently been recognized as an additional independentrisk factor for CDI infection (Issa et al. Clin Gastroenterol Hepatol2007; 5:345-51, Rodemann et al. Clin Gastroenterol Hepatol 2007;5:339-4415). CDI in patients with underlying IBD is associated withincreased severity of colitis and higher rates of recurrence andcolectomy (Issa et al. Inflamm Bowel Dis 2008; 14:1432-42).

It is now recognized that the presence of normal, healthy, intestinalmicrobiota (normal gut microorganisms) offers protection against CDI.Conversely, severe disruption of normal intestinal microbiota by use ofantibiotics, including metronidazole and vancomycin that are used totreat CDI, is likely one of the major reason for its recurrence. Changand colleagues used 16S rDNA sequencing to analyze the fecal microbiotaof seven patients with initial and recurrent CDI (Chang et al. J InfectDis 2008; 197:435-8). They reported that bacterial species diversity wasreduced in all patients compared to normal control subjects. Thegreatest reduction in species diversity, however, was found in the threepatients with recurrent CDI and disruption of their gut microbiota wasevident at the phylum level—with marked reduction in Bacteroidetes,normally one of the two dominant phyla in the colon. Instead, the gutmicrobiota in these patients were dominated by members of theproteobacteria and verrucomicrobia phyla, which normally are only minorconstituents of the colon microbiota.

The general aim of antibiotic treatment for recurrent CDI is not meresuppression of C. difficile, but also preservation of the residual colonmicrobiota and optimization of their restoration. Various antibioticregimens, including long tapered or pulsed dosing with vancomycin(McFarland et al., Am J Gastroenterol 2002; 97:1769-75) and rifaximin“chaser” protocols (Johnson et al. Clin Infect Dis 2007; 44:846-8,Johnson et al. Anaerobe 2009; 15:290-1) have been used to achieve thisobjective with partial success. Recently, fidaxomicin, a new macrocyclicantibiotic which is narrow in spectrum and spares Bacteroides species,was shown to reduce the initial relapse rate of CDI by 50% compared tovancomycin treatment (Louie et al. N Engl J Med 2011; 364:422-31).However, treatment with fidaxomicin did not alter the recurrence rate ofCDI caused by the more virulent PCR 027/NAP1 strain. Therefore, despitethese advances it seems likely that the challenges in treatment ofrecurrent CDI will remain for the foreseeable future.

Fecal microbiota transplantation (FMT), also commonly known as ‘fecalbacteriotherapy’ represents the one therapeutic protocol that allows thefastest reconstitution of a normal composition of colon microbialcommunities. For many decades, FMT has been offered by select centersacross the world, typically as an option of last resort for patientswith recurrent CDI. The mostly commonly earliest cited report for FMTwas by Eiseman and colleagues who in 1958 described the use of fecalenemas for patients who likely had severe or fulminant form ofpseudomembranous colitis (Eiseman et al. Surgery 1958; 44:854-9). Sincethis time, well over 200 cases have been reported as individual casereports, or small case series, with a ˜90% cumulative success rate inclearing recurrent CDI, without any noted adverse events. The historyand general methodology used for FMT have been described in severalrecent reviews (Bakken. Anaerobe 2009; 15:285-9, van Nood et al. EuroSurveill 2009; 14, Khoruts and Sadowsky. Mucosal Immunol 2011; 4:4-7).However, despite the long and successful track record, as well as greatclinical need, the availability of the procedure for many patientsremains very limited.

The lack of wider practice of FMT is due in large part to multiplenon-trivial practical barriers and not due to lack of efficacy. Theseinclude lack of reimbursement for donor screening, lack of adequatedonors at the correct time, difficulty in material preparation andadministration, as well as aesthetic concerns about doing the procedurein endoscopy or medical office. These also include patient perception ofthe procedure, willingness of staff to perform the procedure, sanitationissues related to manipulation of fecal matter. Together these factorsmake it a distasteful option that is often considered a treatment oflast resort, and that is largely unavailable to the vast majority ofpatients who could benefit from it. Moreover, the pharmaceuticalindustry has shown little interest in technological development ofFMT-based therapeutics, in large part due to the wide availability ofdonor material and its complex composition. Instead, development hasbeen driven mostly by individual clinicians faced with desperate need intheir patients.

SUMMARY OF THE INVENTION

The present invention provides compositions that include an extract or apreparation of human feces. In one embodiment, a composition includes nogreater than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% weight non-livingmaterial/weight biological material. Optionally the biological materialincludes human gut, colon or intestinal fecal microbes, and optionallythe biological material includes human gut, colon or intestinalbacteria. Optionally the composition includes a pharmaceuticallyacceptable carrier. Optionally the composition is a formulation for oraladministration.

In one embodiment, a composition consists of, or consists essentiallyof, particles of non-living material and/or particles of biologicalmaterial that will pass through a sieve having a sieve size of 2.0 mm,1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2 mm. Optionally the compositionincludes a pharmaceutically acceptable carrier, and optionally thecomposition is a formulation for oral administration.

In one embodiment, a composition includes at least 4 different phyla ofgut, colon or intestinal bacteria extracted or prepared from the gut,colon or intestine, wherein the phyla include a member of Bacteroidetesphylum, member of Firmicutes phylum, member of Proteobacteria phylum,member of Tenericutes phylum, or a combination thereof. Optionally thephyla are chosen from Bacteroidetes, Firmicutes, Proteobacteria, andTenericutes. The composition includes no greater than 0.05%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, or 10% weight non-living material/weight biological material.Optionally the biological material includes human gut, colon orintestinal flora. Optionally the biological material includes human gut,colon or intestinal bacteria. Optionally the composition includes apharmaceutically acceptable carrier, and optionally the composition is aformulation for oral administration.

In one embodiment, a composition includes an extract of human feceswherein the composition is substantially odorless, optionally includesbiological material, and optionally wherein the biological materialincludes bacteria. Optionally the composition includes apharmaceutically acceptable carrier, and optionally the composition is aformulation for oral administration.

A composition of the present invention may include no greater than 0.1%weight non-living material/weight biological material. In oneembodiment, a composition may consist of, or consist essentially of,particles that will pass through a 0.25 mm sieve, or equivalent. In oneembodiment, a composition may include no greater than 0.05%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%,9% or 10% weight non-living material/weight biological material. Acomposition of the present invention may further include acryoprotectant, such as glycerol. In one embodiment, a composition maybe at a temperature of less than 0° C. In one embodiment, a compositionis a solid, such as a powder. A composition of the present invention mayinclude at least 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, or 5×10¹⁰ bacteria. Inone embodiment, the biological material of a composition may include aplurality of prokaryotic cells, eukaryotic cells, or viruses; or apopulation of prokaryotic cells, eukaryotic cells, and viruses, that issubstantially identical to or representative of or equivalent to apopulation of prokaryotic cells, eukaryotic cells, and viruses presentin a feces of a normal healthy human. In one embodiment, the biologicalmaterial of a composition may include a population of prokaryotic cellsand viruses that is substantially identical to or representative of orequivalent to a population of prokaryotic cells and viruses present inthe feces of a normal healthy human. In one embodiment, the biologicalmaterial of a composition includes a population of prokaryotic cells,eukaryotic cells, or viruses that is substantially identical to orrepresentative of or equivalent to a population of prokaryotic cells,eukaryotic cells, and viruses present in the feces of a normal healthyhuman.

The present invention also provides composition prepared by a process.In one embodiment, a process includes subjecting a fecal sample to acondition or conditions that remove at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more of the non-living material present in thefecal sample. In one embodiment, a process includes filtering a fecalsample with a filter medium, wherein the filter medium includes a sievesize of no greater than 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2 mmto result in or to generate a filtrate. Optionally a compositionincludes a biological material, and optionally the biological materialincludes bacteria. Optionally a composition includes a pharmaceuticallyacceptable carrier. Optionally a composition is a formulation for oraladministration. Optionally the process may occur at a temperature of nogreater than 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33°C., or 34° C.

The composition may include at least 4 different phyla of bacteria,wherein the include a member of Bacteroidetes phylum, member ofFirmicutes phylum, member of Proteobacteria phylum, member ofTenericutes phylum, or a combination thereof. Optionally the phyla arechosen from Bacteroidetes, Firmicutes, Proteobacteria, and Tenericutes.In one embodiment, the composition further includes at least 5, 6, 7, 8,9, or 10 different classes of bacteria chosen from Actinobacteria,Bacteroidia, Bacilli, Clostridia, Erysipelotrichi, Alphaproteobacteria,Betaproteobacteria, Gammaproteobacteria, Mollicutes, andVerrucomicrobiae.

The process may further include adding a cryoprotectant, for instance,glycerol, to the composition. The process may further include freezingthe composition. The composition may be for use as a therapeutic agent,and it may be for use in the treatment of a disease or a pathological oriatrogenic condition of the colon. The disease may be a disease orcondition characterized by a dysfunctional or pathological compositionof colon microbiota, for instance, a Clostridium difficile colitis.

The present invention also provides a method for replacing orsupplementing or modifying a subject's colon microbiota. The method mayinclude administering to the subject a composition described herein. Thepresent invention also provides a method for treating a subject. Themethod may include administering to a subject in need thereof aneffective amount of a composition described herein. The methods mayfurther include removal of some, most, or substantially all of thesubject's colon, gut or intestinal microbiota prior to theadministering. The subject may have or be at risk for having a colitis.In one embodiment, the colitis is an autoimmune colitis, such as aninflammatory bowel disease, an ulcerative colitis, a Crohn's disease, oran irritable bowel syndrome. In one embodiment, the colitis is aninfectious colitis, such as a Clostridium difficile colitis or anenterohemorrhagic colitis. The Clostridium difficile colitis may be anacute Clostridium difficile colitis, a relapsing Clostridium difficilecolitis, and a severe Clostridium difficile colitis. Theenterohemorrhagic colitis may be caused by a Shigella spp. or an E.coli. The subject may have or be at risk for chronic diarrhea or chronicconstipation.

The present invention also provides the use of a composition describedherein for the manufacture of a medicament, or for the manufacture of amedicament for treating or ameliorating or preventing a disease or apathological or iatrogenic condition of the colon. Optionally thedisease is a disease or condition characterized by a dysfunctional orpathological composition of colon microbiota, or the disease is aClostridium difficile colitis, or the disease or condition is a colitis,an autoimmune colitis, an infectious colitis or an enterohemorrhagiccolitis.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of microbial community taxonomic distribution at theorder level in a fecal transplant bacteriotherapy recipient before andafter receiving the transplant in accordance with Example 1.

FIG. 2 is a plot of microbial community taxonomic distribution at thefamily level in a fecal transplant bacteriotherapy recipient before andafter receiving the transplant in accordance with Example 1.

FIG. 3 is a plot of community analysis done using the Yue and Clayton'stheta index showing that the post-transplantation samples from arecipient clustering closely with each other and with the donor'ssample, compared to that of the recipient's pre-transplantation sample.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Before the present invention standard practices suggested matching eachrecipient of fecal bacteriotherapy with a separate donor, usually aclose family member, or using the recipient's own banked feces for lateruse. The rationale for these practices was the idea that close familymembers have already shared their pathogens, and that these kinds of gutmicrobiota would be somehow better tolerated by the recipient's immunesystem because of previous exposure. However, this resulted induplicative screening, burdening already debilitated patients with thetask of finding a suitable donor, pressure on the donor to provide thematerial and potentially withholding important medical information,pressure to decrease costs since costs were usually borne by thepatient, time delays associated with the screening, and pressure toaccept donors of suboptimal health status during donor selection. Thecompositions presented herein result from a more standardizedmanufacturing process with rigorous donor screening, multiple steps offiltration that concentrate the microbiota and remove the bulk ofnonliving material, and optionally freeze/thaw it in a way thatpreserves its viability. The compositions presented herein provide asignificant advantage by making useful compositions of colon microflorareadily available for use by a physician to treat a patient. Moreover,it is much more aesthetically acceptable, as the compositions are nearlyodorless, are in concentrated form, and are easily manipulated usingstandard laboratory practice.

The present invention provides compositions that include fecal microbes.As used herein, the term “fecal microbes” refers to microorganisms thatare present in the gut, intestine, or colon, preferably colon, of anormal healthy adult human. Such a composition may be prepared byprocessing fecal material. As used herein, the term “fecal material”refers to human stool. Unprocessed fecal material contains non-livingmaterial and biological material. The “non-living material” may include,but is not limited to, dead bacteria, shed host cells, proteins,carbohydrates, fats, minerals, mucus, bile, undigested fiber and otherfoods, and other compounds resulting from food and metabolic wasteproducts and partial or complete digestion of food materials.“Biological material” refers to the living material in fecal material,and includes microbes including prokaryotic cells such as bacteria andarchea (e.g., living prokaryotic cells and spores that can sporulate tobecome living prokaryotic cells), eukaryotic cells such as protozoa andfungi, and viruses. In one embodiment, “biological material” refers tothe living material, e.g., the microbes, eukaryotic cells, and viruses,that are present in the colon of a normal healthy human.

Examples of prokaryotic cells that may be present in a composition ofthe present invention include cells that are members of the classActinobacteria, such as the subclass Actinobacteridae orCoriobacteridae, such as the order Bifidobacteriales orCoriobacteriales, and/or such as the family Bifidobacteriaceae orCoriobacteriaceae; members of the phylum Bacteroidetes, such as classBacteroidia, such as class Bacteroidales, and/or such as familyBacteroidaceae or Rikenellaceae; members of the phylum Firmicutes, suchas class Bacilli, Clostridia, or Erysipelotrichi, such as orderBacillales or Lactobacillales or Clostridales or Erysipelotrichales,and/or such as family Paenibacillaceae or Aeroccaceae orLactobacillaceae or Streptococcaceae or Catabacteriaceae orPeptococcaceae or Peptostreptococcaceae or Ruminococcaceae orClostridiaceae or Eubacteriaceae or Lachnospiraceae orErysipelotrichaceae; members of the phylum Proteobacteria, such as classAlphaproteobacteria or Betaproteobacteria or Gammaproteobacteria, suchas order Rhizobiales or Burkholderiales or Alteromonadales orEnterobacteriales, and/or such as family Rhodobiaceae orBurkholderiaceae or Shewanellaceae or Enterobacteriaceae; members of thephylum Tenericutes, such as the class Mollicutes, such as the orderEntomoplasmatales, and/or such as the family Spiroplasmataceae; and/ormembers of the class Verrucomicrobiae, such as the orderVerrucomicrobiales, and/or such as the family Verrucomicrobiaceae.

In one embodiment a composition of the present invention may includeprokaryotic bacteria that are members of at least 1 phylum, at least 2phyla, at least 3 phyla, at least 4 phyla, at least 5 phyla, at least 6phyla, at least 7 phyla, at least 8 phyla, at least 9 phyla, or at least10 phyla. In one embodiment a composition of the present invention mayinclude prokaryotic bacteria that are members of at least 1 class, atleast 2 classes, at least 3 classes, at least 4 classes, at least 5classes, at least 6 classes, or at least 7 classes. In one embodiment acomposition of the present invention may include prokaryotic bacteriathat are members of at least 1 order, at least 2 orders, at least 3orders, at least 4 orders, at least 5 orders, at least 6 orders, or atleast 7 orders. In one embodiment a composition of the present inventionmay include prokaryotic bacteria that are members of at least 1 family,at least 2 families, at least 3 families, at least 4 families, at least5 families, at least 6 families, at least 7 families. In one embodimenta composition of the present invention may include at least 5, at least10, at least 20, or at least 30 different genera of prokaryoticbacteria. In one embodiment a composition of the present invention mayinclude at least 10, at least 50, at least 100, at least 200, at least300, or at least 400 different species of prokaryotic bacteria.

In one embodiment a composition of the present invention includes nogreater than 5% weight of non-living material/weight biological material(wt/wt), no greater than 2.5% (wt/wt), no greater than 1% (wt/wt), nogreater than 0.1% (wt/wt), no greater than 0.01% (wt/wt), or no greaterthan 0.001% (wt/wt) nonliving material. In one embodiment, the amount ofnon-living material in a composition of the present invention isundetectable using currently available techniques. For instance, livingmaterial can be stained for biological activity, electron transport, DNAand RNA for specific genes.

In one embodiment, the fecal material present in a composition of thepresent invention does not include particles (e.g., particles ofnon-living material and/or particles of biological material) having asize of greater than 2.0 millimeters (mm), greater than 1.0 mm, greaterthan 0.5 mm, greater than 0.25 mm, greater than 0 212 mm, greater than0.180 mm, greater than 0.150 mm, greater than 0.125 mm, greater than0.106 mm, greater than 0.090 mm, greater than 0.075 mm, greater than0.063 mm, greater than 0.053 mm, greater than 0.045 mm, greater than0.038 mm, greater than 0.032 mm, greater than 0.025 mm, greater than0.020 mm, greater than 0.01 mm, or greater than 0.2 mm. Non-fecalmaterial present in a composition may include particles having a size ofgreater than 2.0 mm, greater than 1.0 nun, greater than 0.5 mm, greaterthan 0.25 mm, greater than 0.212 mm, greater than 0.180 mm, greater than0.150 mm, greater than 0.125 mm, greater than 0.106 mm, greater than0.090 mm, greater than 0.075 mm, greater than 0.063 mm, greater than0.053 mm, greater than 0.045 mm, greater than 0.038 mm, greater than0.032 mm, greater than 0.025 mm, greater than 0.020 mm, greater than0.01 mm, or greater than 0.2 mm. In one embodiment, the fecal materialpresent in a composition of the present invention consists of, orconsists essentially of, particles of non-living material and/orbiological material having a size that will pass through a sieve havinga sieve size of 2.0 nun, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm,0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm,0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2 mm.Thus, in such an embodiment, the fecal material present in a compositionhas a size that is less than or equal to 2.0 mm, less than or equal to1.0 mm, less than or equal to 0.5 mm, less than or equal to 0.25 mm,less than or equal to 0.212 mm, less than or equal to 0.180 mm, lessthan or equal to 0.150 mm, less than or equal to 0.125 mm, less than orequal to 0.106 mm, less than or equal to 0.090 mm, less than or equal to0.075 mm, less than or equal to 0.063 mm, less than or equal to 0.053mm, less than or equal to 0.045 mm, less than or equal to 0.038 mm, lessthan or equal to 0.032 mm, less than or equal to 0.025 mm, less than orequal to 0.020 mm, less than or equal to 0.01 mm, or less than or equalto 0.2 mm. The sieve size may be based on the US Standard sieve sizesof, for instance, 10, 18, 35, 60, 70, 80, 100, 120, 140, 170, 200, 230,270, 325, or 400.

A composition of the present invention may optionally include acryoprotectant. A cryoprotectant is a compound that maintains theviability of fecal microbes when frozen. Cryoprotectants are known inthe art and used routinely to protect microbes when exposed to freezingconditions. Examples include, but are not limited to, amino acids suchas alanine, glycine, proline; simple sugars such as sucrose, glucose,lactose, ribose, and trehalose; and other compounds such as dimethylsulfoxide (DMSO), and glycerol. The amount of cryoprotectant present ina composition described herein may vary depending on the cryoprotectantused and the temperature to be used for freezing (e.g., −20° C., −80°C., or a different temperature). The amount of cryoprotectant that canbe used is known to the skilled person or may be easily determined usingroutine experimentation. In one embodiment, a composition of the presentinvention may include glycerol at a concentration of 10%.

In one embodiment a composition of the present invention does notinclude pathogenic biological material. In one embodiment, fecalmaterial is from a person that has undergone a medical history, aphysical examination, and laboratory testing. The evaluation of medicalhistory may include, but is not limited to, risk of infectious agents,presence of gastrointestinal co-morbidities, factors that can or doaffect the composition of the intestinal microbiota, and systemicmedical conditions. Exclusion criteria regarding risk of infectiousagents may include, but are not limited to, known viral infection withHepatitis B, C or HIV; known exposure to HIV or viral hepatitis at anytime; high risk behaviors including sex for drugs or money, men who havesex with men, more than one sexual partner in the preceding 12 months,any past use of intravenous drugs or intranasal cocaine, history ofincarceration; tattoo or body piercing within 12 months; travel to areasof the world where risk of traveler's diarrhea is higher than the US;and current communicable disease, e.g., upper respiratory viralinfection.

Exclusion criteria regarding gastrointestinal co-morbidities include,but are not limited to, history of irritable bowel syndrome, whereinspecific symptoms may include frequent abdominal cramps, excessive gas,bloating, abdominal distension, fecal urgency, diarrhea, constipation;history of inflammatory bowel disease such as Crohn's disease,ulcerative colitis, microscopic colitis; chronic diarrhea; chronicconstipation or use of laxatives; history of gastrointestinal malignancyor known colon polyposis; history of any abdominal surgery, e.g.,gastric bypass, intestinal resection, appendectomy, cholecystectomy, andthe like; use of probiotics or any other over the counter aids used bythe potential donor for purpose of regulating digestion, but yogurt andkefir products may be allowed if taken merely as food rather thannutritional supplements.

Exclusion criteria regarding factors that can or do affect thecomposition of the intestinal microbiota include, but are not limitedto, antibiotics for any indication within the preceding 6 months; anyprescribed immunosuppressive or anti-neoplastic medications.

Exclusion criteria regarding systemic medical conditions include, butare not limited to, established or emerging metabolic syndrome, wherecriteria used for definition here are stricter than establishedcriteria, including history of increased blood pressure, history ofdiabetes or glucose intolerance; known systemic autoimmunity, e.g.,connective tissue disease, multiple sclerosis; known atopic diseasesincluding asthma or eczema; chronic pain syndromes includingfibromyalgia, chronic fatigue syndrome; ongoing (even if intermittent)use of any prescribed medications, including inhalers or topical creamsand ointments; neurologic, neurodevelopmental, and neurodegenerativedisorders including autism, Parkinson's disease.

Exclusion criteria on physical examination may include, but are notlimited to, general, such as body mass index >26 kg/m², central obesitydefined by waste:hip ratio >0.85 (male) and >0.80 (female); bloodpressure >135 mmHg systolic and >85 mmHg diastolic; skin—presence of arash, tattoos or body piercing placed within a year, jaundice; enlargedlymph nodes; wheezing on auscultation; hepatomegaly or stigmata of liverdisease; swollen or tender joints; muscle weakness; abnormal neurologicexamination.

Exclusion criteria on laboratory testing may include, but is not limitedto, positive stool Clostridium difficile toxin B tested by PCR; positivestool cultures for any of the routine pathogens including Salmonella,Shigella, Yersinia, Campylobacter, E. coli 0157:H7; abnormal ova andparasites examination; positive Giardia, Cryptosporidium, orHelicobacter pylori antigens; positive screening for any viralillnesses, including HIV 1 and 2, Viral Hepatitis A IgM, Hepatitissurface antigen and core Ab; abnormal RPR (screen for syphilis); anyabnormal liver function tests including alkaline phosphatase, aspartateaminotransaminase, alanine aminotransferase; raised serumtriglycerides >150 mg/dL; HDL cholesterol <40 mg/dL (males) and <50mg/dL (females); high sensitivity CRP >2.4 mg/L; raised fasting plasmaglucose (>100 mg/dL).

The compositions of the present invention may be included in a diversityof pharmaceutically acceptable formulations. In one embodiment, aformulation may be a fluid composition. Fluid compositions include, butare not limited to, solutions, suspensions, dispersions, and the like.In one embodiment, a formulation may be a solid composition. Solidcompositions include, but are not limited to, powder, granule,compressed tablet, pill, capsule, chewing gum, wafer, and the like.Those formulations may include a pharmaceutically acceptable carrier torender the composition appropriate for administration to a subject. Asused herein “pharmaceutically acceptable carrier” includespharmacologically inactive compounds compatible with pharmaceuticaladministration. The compositions of the present invention may beformulated to be compatible with its intended route of administration. Acomposition of the present invention may be administered by any methodsuitable for depositing in the gastrointestinal tract, preferably thecolon, of a subject. Examples of routes of administration include rectaladministration (e.g., by suppository, enema, upper endoscopy, upper pushenteroscopy, or colonoscopy), intubation through the nose or the mouth(e.g., by nasogastric tube, nasoenteric tube, or nasal jejunal tube), ororal administration (e.g., by a solid such as a pill, tablet, orcapsule, or by liquid).

For therapeutic use in the method of the present invention, acomposition may be conveniently administered in a form containing one ormore pharmaceutically acceptable carriers. Suitable carriers are wellknown in the art and vary with the desired form and mode ofadministration of the composition. For example, they may includediluents or excipients such as fillers, binders, wetting agents,disintegrators, surface-active agents, glidants, lubricants, and thelike. Typically, the carrier may be a solid (including powder), liquid,or combinations thereof. Each carrier is preferably “acceptable” in thesense of being compatible with the other ingredients in the compositionand not injurious to the subject. The carrier is preferably biologicallyacceptable and inert, i.e., it permits the composition to maintainviability of the biological material until delivered to the appropriatesite.

Oral compositions may include an inert diluent or an edible carrier. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules, e.g., gelatin capsules. Oral compositions can alsobe prepared by combining a composition of the present invention with afood. In one embodiment a food used for administration is chilled, forinstance, ice cream. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

The active compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from, for instance, Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art.

In one embodiment, a composition may be encapsulated. For instance, whenthe composition is to be administered orally, the dosage form isformulated so the composition is not exposed to conditions prevalent inthe gastrointestinal tract before the colon, e.g., high acidity anddigestive enzymes present in the stomach and/or intestine. Theencapsulation of compositions for therapeutic use is routine in the art.Encapsulation may include hard-shelled capsules, which may be used fordry, powdered ingredients soft-shelled capsules. Capsules may be madefrom aqueous solutions of gelling agents such as animal protein (e.g.,gelatin), plant polysaccharides or derivatives like carrageenans andmodified forms of starch and cellulose. Other ingredients may be addedto a gelling agent solution such as plasticizers (e.g., glycerin and orsorbitol), coloring agents, preservatives, disintegrants, lubricants andsurface treatment.

A composition may be prepared by obtaining a fecal sample from anappropriate donor and blending with a diluent. Useful diluents includeaqueous solutions that are routinely used for manipulating microbes,eukaryotic cells, and/or viruses. Useful diluents may includeconstituents to maintain physiological buffer, osmolarity, and the like.The diluent is preferably sterile and/or non-allergenic. An example of adiluent includes, but is not limited to, phosphate buffered saline at pH7. In one embodiment, 1 part donor feces may be combined with 5 partsdiluent (e.g., 50 grams of donor feces may be combined with 250 mlsdiluent) and blended. In one embodiment, the oxygen in the blendingchamber may be decreased or removed by purging with an inert gas such asnitrogen or argon prior to blending. Such anaerobic conditions may beuseful to maintain viability of most anaerobic bacteria present in acolon. The sample may be blended multiple times and/or more diluent maybe added until a consistency is achieved that will permit the followingsteps to occur. In one embodiment, anaerobic conditions are not used insteps following the blending. It was found that anaerobic conditionswere not necessary in the steps following the blending, and this wasunexpected and surprising since a substantial percentage of prokaryoticcells in fecal material are strict anaerobes, and exposure to oxygenkills them. After the blending, the solutions used for washing andresuspension did not need to be purged of oxygen, and manipulation ofthe microbiota in an oxygen-free cabinet or glove box was not needed.

Not all microbes and eukaryotic cells present in an individual's coloncan be cultured, thus, in one embodiment conditions for preparing acomposition include the use of temperatures that decrease thereplication of the microbes and eukaryotic cells. In one embodiment, theconditions used for preparation are maintained below 37° C. Forinstance, the conditions used for preparation are maintained at atemperature of no greater than 30° C., no greater than 20° C., nogreater than 10° C., or no greater than 5° C. In one embodiment,conditions are used such that replication of the microbes and eukaryoticcells does not occur. When the conditions used to prepare a compositionof the present invention include lower temperatures to minimizereplication and cell death, the biological material present in acomposition includes a population of microbes, eukaryotic cells, andviruses that is essentially identical to a population of microbes,eukaryotic cells, and viruses present in the colon or feces of a normalhealthy human, e.g., the donor from whom the fecal sample was obtained.

Removal of non-living material may be achieved by passing the blendedsample through a sieve with a sieve size of no greater than 2.0 mm, nogreater than 1.0 mm, no greater than 0.5 min, no greater than 0.25 mm,no greater than 0.212 mm, no greater than 0.180 mm, no greater than0.150 mm, no greater than 0.125 mm, no greater than 0.106 mm, no greaterthan 0.090 mm, no greater than 0.075 mm, no greater than 0.063 mm, nogreater than 0.053 mm, no greater than 0.045 mm, no greater than 0.038mm, no greater than 0.032 mm, no greater than 0.025 mm, no greater than0.020 nun, no greater than 0.01 mm, or no greater than 0.2 mm. In oneembodiment, the blended sample is prepared by passing it through a sievewith a sieve size of 0.25 mm and collecting the filtrate. In oneembodiment, the blended sample is passed through sieves withprogressively smaller sieve sizes until final passage through a sievesize of 0.25 mm. For instance, if a total of four sieves are used thesieve size of the first sieve may be 2 mm, followed by 1 mm, followed by0.5 mm, and then followed by 0.25 mm. The final filtrate may becollected in a centrifuge tube, and centrifuged at a speed sufficient topellet the biological material, for instance, 10,000×g for 10 minutes at4° C. The supernatant is removed, the cells are resuspended in diluent,optionally centrifuged again, for instance at 10,000×g for 10 minutes at4° C. The final supernatant is discarded and the cells are resuspendedin an aqueous solution (e.g., diluent, cryoprotectant, and the like, ora combination thereof). In one embodiment, the volume of the blendedmixture is decreased through the steps of sieving and washing. Forinstance, in one embodiment, the volume is decreased to 14% of thevolume used in the blending (e.g., from 250 mls to 35 mls). In oneembodiment, the volume of the blended mixture is decreased through thesteps of sieving and washing to result in between 1×10¹⁰ and 5×10¹⁰cells in a volume that is subsequently administered to a subject. Thisprocess results in an extract of feces that is highly enriched for allcolon microbiota that are able to pass through a sieve as describedabove, and can be centrifuged at 10,000×g for 10 minutes. As usedherein, “enriched” refers to increasing the abundance of biologicalmaterial relative to non-living material, such that biological materialconstitutes a significantly higher proportion compared to the fecalmaterial before the enrichment. The term “enriched” refers to thosesituations in which a person has intervened to elevate the proportion ofbiological material.

The amount of aqueous solution added may be in an amount to result in asingle dosage having an appropriate number of cells. In one embodiment,a single dosage may include between 1×10¹⁰ and 5×10¹⁰ cells, forinstance, 3×10¹⁰ cells. Since most biological material is difficult orimpossible to culture, a hemocytometer may be used to determine thenumber of cells.

In one embodiment the resulting pellet may be suspended in half theoriginal volume of diluent containing 10% glycerol. The sample may beused immediately, or may be frozen, for instance, at −80° C., for lateruse. When freezing, the sample may be left in a centrifuge tube, or maybe in a different container. In one embodiment, the container is onethat increases the surface area of the sample. For instance, the samplemay be placed in an IV bag. When the frozen sample is to be used, it maybe thawed on ice and then transplanted into the recipient. It was foundthat freezing the compositions described herein did not result indestruction of its curative potential. In one embodiment the sampleresulting from centrifugation may be processed for long term storage of1 year or longer. The ability to store such a sample provides a level offlexibility that was not possible with other methods. For instance, itwas necessary to quickly identify a donor, rapidly process a fecalsample from the donor, and use it immediately. Examples of usefulprocessing methods include, but are not limited to, freezing, freezedrying, spray drying, lyophilization, vacuum drying, air drying, orother forms of evaporative drying. Processing of a composition of thepresent invention may include the production of a powder following anydrying procedure.

The use of sieves to extract biological material from fecal materialunexpectedly resulted in a composition which was nearly odorless. Thiswas not expected because feces normally have a distinctive odor and thiswas surprising to be removed by the minimal manipulation used. This is asignificant advantage as it takes a method that is unaesthetic and sodistasteful that some patients and staff refuse to take part, andchanges it into a method that is easily practiced in a normal clinicalsetting or at home. As used herein, “odorless” means there is adecreased amount of volatile organic molecules present, and thedecreased amount of volatile organic molecules present can be easilydetected by a person comparing the material before processing with thematerial after processing.

The present invention is further directed to methods of using thecompositions described herein. A method of the present inventionincludes administering to a subject in need thereof an effective amountof a composition described herein. The administering is under conditionssuitable for deposition of the composition in a region of the large orsmall intestine such that the biological material in the compositioncolonizes the colon. For instance, administration may be into uppergastrointestinal tract, as well as lower gastrointestinal tract, e.g.,the terminal ileum, cecum, colonic areas containing diverticulosis, andrectum. In one embodiment the administering may be oral, such as bytablet. In one embodiment the administering may be by intubation, suchas by nasogastric tube. In one embodiment the administering may berectal, for instance by a colonoscope, enema, or suppository. Conditionsthat are “suitable” for an event to occur, or “suitable” conditions areconditions that do not prevent such events from occurring. Thus, theseconditions permit, enhance, facilitate, and/or are conducive to theevent. As used herein, an “effective amount” relates to a sufficientamount of a composition described herein, to provide the desired effect.For instance, in one embodiment an “effective amount” is an amounteffective to alleviate one or more symptoms and/or signs of the diseaseas described herein. In some embodiments, an effective amount is anamount that is sufficient to effect a reduction in a symptom and/or signassociated with a disease, such as diarrhea or C. difficile. A reductionin a symptom and/or a sign is, for instance, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 100% in a measured sign as comparedto a control, a non-treated subject, or the subject prior toadministration of the composition. In one embodiment, an effectiveamount is an amount sufficient to result in at least 1×10¹⁰, at least3×10¹⁰, or at least 5×10¹⁰ cells delivered to the colon. It will beunderstood, however, that the total dosage of the compositions asdisclosed herein will be decided by the attending physician within thescope of sound medical judgment. The exact amount required will varydepending on factors such as the type and extent of disease beingtreated.

In one embodiment, a method of the present invention includes treatingcertain diseases in a subject in need of treatment. The subject may be amammal, such as a human. In some embodiments animal models may be used,such as a mammal, including a rat, a mouse, a hamster, a gerbil, or aprimate. As used herein, the term “disease” refers to any deviation fromor interruption of the normal structure or function of a part, organ, orsystem, or combination thereof, of a subject that is manifested by acharacteristic symptom or clinical sign. Diseases include thosecharacterized by dysfunctional composition of colon microbiota. Suchdiseases include, but are not limited to, colitis, including autoimmunecolitis (e.g., inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, irritable bowel syndrome) and infectious colitis. Examples ofinfectious colitis include, but are not limited to Clostridium difficilecolitis (e.g., acute C. difficile colitis, relapsing C. difficilecolitis, or severe C. difficile colitis) and enterohemorrhagic colitis(e.g., a colitis caused by Shigella spp. or E. coli). Other examples ofdiseases include, but are not limited to, chronic diarrhea; chronicconstipation, metabolic syndrome and obesity, atopic diseases includingasthma, eczema, eosinophilic disorders of the GI tract, systemicautoimmunity including rheumatoid arthritis, systemic lupuserythematosis, multiple sclerosis, etc., chronic pain disorders suchfibromyalgia, chronic fatigue syndrome, neurodegenerative disorders,eating disorders, and malnutrition.

As used herein, the term “symptom” refers to subjective evidence ofdisease or condition experienced by the patient and caused by disease.As used herein, the term “clinical sign,” or simply “sign,” refers toobjective evidence of a disease present in a subject. Symptoms and/orsigns associated with diseases referred to herein and the evaluation ofsuch signs are routine and known in the art. Typically, whether asubject has a disease, and whether a subject is responding to treatment,may be determined by evaluation of signs associated with the disease.

Treatment of a disease can be prophylactic or, alternatively, can beinitiated after the development of a disease. Treatment that isprophylactic, for instance, initiated before a subject manifests signsof a disease, is referred to herein as treatment of a subject that is“at risk” of developing a disease. An example of a subject that is atrisk of developing a disease is a person having a risk factor. Anexample of a risk factor for Clostridium difficile colitis is antibiotictherapy of the gastrointestinal tract. Treatment can be performedbefore, during, or after the occurrence of the diseases describedherein. Treatment initiated after the development of a disease mayresult in decreasing the severity of the signs of the disease, orcompletely removing the signs.

In one embodiment, a method of the present invention includestransplanting a microbiota from a donor to a recipient.

In one embodiment, a method of the present invention includes increasingthe relative abundance of members of the phylum Firmicutes, such as anonpathogenic member of the class Clostridia, and/or members of thephylum Bacteroidetes, in a recipient's colon. The phrase “relativeabundance” refers to number of members of a phylum or class compared tothe number of members of all other taxa in a recipient's colon. Such acomparison can be expressed as a percent. In one embodiment, therelative abundance of non-pathogenic members of the class Clostridia ina recipient's colon after the administration may be increased by atleast 5%, at least 10%, at least 20%, or at least 50%, compared to therecipient's colon before the administration. In one embodiment, therelative abundance of members of the phylum Firmicutes in a recipient'scolon after the administration may be increased by at least 5%, at least10%, at least 20%, or at least 50% compared to the recipient's colonbefore the administration. The change in the abundance may be determinedat, for instance, 3 days, 10 days, 15 days, or 25 days after theadministration of fecal microbiota.

In one embodiment, a method of the present invention includes decreasingthe relative abundance of members of the phylum Proteobacteria in arecipient's colon. In one embodiment, the relative abundance of membersof the phylum Proteobacteria in a recipient's colon after theadministration may be decreased by at least 10%, at least 20%, at least30%, or at least 40% compared to the recipient's colon before theadministration. The change in the abundance of members of the phylumProteobacteria may be determined at, for instance, 3 days, 10 days, 15days, or 25 days after the administration.

In one embodiment, the existing microbiota does not need to be clearedprior to administration of a composition of the present invention. Inother embodiments clearance of the microbiota may be necessary. Methodsfor clearance of existing microbiota are known and routine. In oneexample, clearance can be accomplished by administering a cocktail ofantibiotics for one week until a day prior to transplant. An example ofa useful cocktail is Metronidazole (1000 mg twice daily), Rifaximin (550mg twice daily), Vancomycin (500 mg twice daily), and Neomycin (1000 mgtwice daily).

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1

Clostridium difficile associated disease is a major known complicationof antibiotic therapy. The pathogen is normally held in check by nativecolon microbiota, but this level of protection may be lost when thesemicrobial communities are suppressed by antibiotics. Antibiotics used totreat C. difficile infection may also perpetuate its recurrence bycontinued suppression of normal microbiota. Thus, a significant fractionof patients suffer from recalcitrant C. difficile infection, andrecalcitrant C. difficile infection is associated with significantmorbidity. Fecal bacteriotherapy is an increasingly used method used tobreak the cycle of C. difficile infection recurrence presumably throughrestoration of normal intestinal microbial communities. We previouslyreported, in one clinical case, that bacteriotherapy of colon microbiotaresulted in the replacement of a host's microbiota by that of the donor(Khoruts, et al., 2010, J. Clin. Gastroenterol., 44(5):354). In order toobtain a greater understanding of the composition and stability ofmicrobial communities before and after bacteriotherapy, we have analyzedamplified 16S rRNA regions of fecal DNA (V5 and V6) by using apyrosequencing technology (an Illumina HiSeq2000 or other Illuminaplatforms). Additional individuals are currently being processed andanalyzed.

Introduction

-   -   Clostridium difficile is an emerging pathogen and the most        common cause of nosocomial diarrhea.    -   Infections are often associated with antibiotic therapy, where        the protective effect provided by the normal intestinal flora is        disrupted.    -   C. difficile infection is often controlled by additional        antimicrobial therapy, but approximately 20% of patients develop        refractory disease resulting in recurrent diarrhea.    -   Bacteriotherapy, in the form of a fecal transplantation, has        been shown to successfully treat refractory C. difficile        infection.    -   Next generation sequencing technologies have allowed for a        deeper interrogation of the intestinal microflora and was used        in our study to examine changes in microbial community structure        after transplantation.

Donor fecal material was obtained from the patient's son, who was testedfor infectious disease, including C. difficile, Hepatitis A, B, or Cviruses, HIV virus, Salmonella, Campylobacter, Yersinia, Shigella, E.Coli 0157:H7, Helicobacter pylori, Treponema pallidum, Giardia, andCryptosporidium.

The patient was infused with donor fecal material by colonoscopy, whichrevealed severe, extensive diverticulosis in the sigmoid colon. Thedonor's fecal material was deposited into the cecum. Symptoms consistentwith C. difficile infection were resolved within days ofbacteriotherapy.

Methods

-   -   Patient fecal samples were collected at day −31 before the fecal        transplant bacteriotherapy and at days 5, 21, 46, 95, 132, 159,        188, and 227 post transplantation. A donor fecal sample was        collected the day of the procedure and deposited into the        recipient's cecum.    -   DNA was extracted from fecal materials using a MOBIO ultra-clean        fecal DNA kit (MOBIO Laboratories, Inc., Carlsbad, Calif.) as        directed by the manufacturer. Triplicate samples were extracted        and pooled.    -   The V6 hypervariable region of the bacteria 16S rRNA gene was        amplified using 50 ng of extracted DNA as template. Barcoded        primers were used for multiplex sequencing (Kysela et al., 2005,        Environmental Microbiology 7:356-64, and Claesson et al.,        Nucleic Acids Research, 2010, Vol. 38, No. 22 e200        doi:10.1093/narigkq873). Triplicate samples were prepared and        pooled.    -   Amplified samples were mixed in equimolar ratios and sequenced        at the National Center for Genomic Research (NCGR) using the        Illumina sequencing platform.    -   Sequence data was analyzed using MOTHUR and the SILVA reference        database (Scholss, 2009, Appl. Environ. Microbiol.,        75(23):7537-7541. The taxonomy of operational taxonomic units        (OTUs) were assigned at the 97% similarity using the GreenGenes        reference files. (FIGS. 1 and 2)    -   Principal component analysis was done using Yue and Clayton's        Theta calculation (Yue and Clayton, 2005, Commun. Stat. Theor.        Methods, 34:2123-2131). Accumulation curves were calculated        based on 97% OTU similarities. (FIG. 3)

Results & Discussion

-   -   Greater than 40% of the sequences obtained from the recipient's        pretransplantation sample (day −31) belonged to unclassified        Mollicutes strains or the Gammaproteobacteria.    -   In contrast, the donor's and recipient's post-transplantation        samples were dominated by Firmicutes. Unclassified members of        the Clostridiales and the Ruminococcaceae family were abundant.    -   Community analysis done using the Yue and Clayton's theta index        showed that the post-transplantation samples clustered more        closely with each other and with the donor sample, compared to        that of the recipient's pre-transplantation sample.    -   Sequence analysis indicated that the taxa present in the        recipient's pre- and post-transplant fecal samples differed        considerably, suggesting that fecal bacteriotherapy was        successful in altering the patient's intestinal microflora.    -   The transplanted microbial community in the recipient's        intestine remained fairly stable after 7.5 months post        transplantation.

Surprisingly, sequences representing the Bacteroidales were in fairlylow abundance in all of the samples analyzed.

TABLE 1 Pyrosequencing Metrics Sample Day Seqs OTUs (97%) CoverageShannon Index −31 1335704 15600 0.992 3.844 Donor 2892413 34945 0.9924.663 +5 2631872 28948 0.992 4.004 +21 2909055 30038 0.993 4.047 +463604923 35864 0.993 4.277 +95 2216996 27782 0.991 4.631 +132 292780026510 0.994 3.746 +159 2691936 30990 0.992 4.551 +188 1649565 184170.993 3.781 +277 3073102 31162 0.993 3.852

Example 2

Donor Screening for Fecal Microbiota Material Preparation

The donor undergoes a complete medical history and physical examination.In addition, a full-length donor history questionnaire is completed asrecommended by the FDA for blood donors, and potential donors saying yesto any of the questions are excluded(http://wwvv.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/UCM213552.pdf).However, as gut microbiota have been associated or postulated to beinvolved with multiple medical conditions, the process of selection ismore rigorous than that of the blood donors and includes virtually anysystemic illness.

Inclusion Criteria

1. Age >18

2. Ability to provide informed consent.

Exclusion Criteria

I. Medical History

-   -   A. Risk of infectious agent.        -   1. Known viral infection with Hepatitis B, C or HIV        -   2. Known exposure to HIV or viral hepatitis at any time        -   3. High risk behaviors including sex for drugs or money, men            who have sex with men, more than one sexual partner in the            preceding 12 months, any past use of intravenous drugs or            intranasal cocaine, history of incarceration.        -   4. Tattoo or body piercing within 12 months.        -   5. Travel to areas of the world where risk of traveler's            diarrhea is higher than the US.        -   6. Current communicable disease, e.g., upper respiratory            viral infection.    -   B. Gastrointestinal comorbidities.        -   1. History of irritable bowel syndrome. Specific symptoms            may include frequent abdominal cramps, excessive gas,            bloating, abdominal distension, fecal urgency, diarrhea,            constipation.        -   2. History of inflammatory bowel disease such as Crohn's            disease, ulcerative colitis, microscopic colitis.        -   3. Chronic diarrhea.        -   4. Chronic constipation or use of laxatives.        -   5. History of gastrointestinal malignancy or known colon            polyposis.        -   6. History of any abdominal surgery, e.g., gastric bypass,            intestinal resection, appendectomy, cholecystectomy, etc.        -   7. Use of Probiotics or any other over the counter aids used            by the potential donor for purpose of regulating digestion.            Yogurt and kefir products are allowed if taken merely as            food rather than nutritional supplements.    -   C. Factors that can or do affect the composition of the        intestinal microbiota.        -   1. Antibiotics for any indication within the preceding 6            months.        -   2. Any prescribed immunosuppressive or anti-neoplastic            medications.    -   D. Systemic Medical Conditions.        -   1. Metabolic Syndrome, established or emerging. Criteria            used for definition here are stricter than any established            criteria. These include history of increased blood pressure,            history of diabetes or glucose intolerance.        -   2. Known systemic autoimmunity, e.g., connective tissue            disease, multiple sclerosis.        -   3. Known atopic diseases including asthma or eczema.        -   4. Chronic pain syndromes including fibromyalgia, chronic            fatigue syndrome.        -   5. Ongoing (even if intermittent) use of any prescribed            medications, including inhalers or topical creams and            ointments.        -   6. Neurologic, neurodevelopmental, and neurodegenerative            disorders including autism, Parkinson's disease.            II. Exclusion Criteria on Physical Examination.    -   1. General. Body mass index >26 kg/m², central obesity defined        by waste:hip ratio >0.85 (male) and >0.80 (female).    -   2. Blood pressure >135 mmHg systolic and >85 mmHg diastolic.    -   3. Skin—presence of a rash, tattoos or body piercing placed        within a year, jaundice.    -   4. Enlarged lymph nodes.    -   5. Wheezing on auscultation.    -   6. Hepatomegaly or stigmata of liver disease.    -   7. Swollen or tender joints. Muscle weakness.    -   8. Abnormal neurologic examination.        III. Exclusion Criteria on Laboratory Testing.    -   1. Positive stool Clostridium difficile toxin B tested by PCR.    -   2. Positive stool cultures for any of the routine pathogens        including Salmonella, Shigella, Yersinia, Campylobacter, E. coli        0157:H7.    -   3. Abnormal ova and parasites examination.    -   4. Positive Giardia, Cryptosporidium, or Helicobacter pylori        antigens.    -   5. Positive screening for any viral illnesses, including HIV 1        and 2, Viral Hepatitis A IgM, Hepatitis surface antigen and core        Ab.    -   6. Abnormal RPR (screen for syphilis).    -   7. Any abnormal liver function tests including alkaline        phosphatase, aspartate aminotransaminase, alanine        aminotransferase.    -   8. Raised serum triglycerides >150 mg/dL    -   9. BDL cholesterol <40 mg/dL (males) and <50 mg/dL (females)    -   10. High sensitivity CRP >2.4 mg/L    -   11. Raised fasting plasma glucose (>100 mg/dL)

Example 3

Fecal Sample Processing

Donor fecal material is immediately chilled on ice for transport to thelaboratory. Samples are processed within one hour after collection.

Fecal samples are homogenized by mixing 50 g of donor feces and 250 mlof sterile phosphate buffered saline, pH 7, (PBS) in a Waring Blender.The blending chamber is purged with nitrogen gas for several minutes toremove oxygen prior to homogenization. Samples are blended three timeson the lowest setting for 20 seconds. Additional PBS or blending cyclesmay be added depending on the consistency of the fecal suspension.Blended samples are passed through a series of four sieves with poresizes of 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm (W.S. Tyler IndustrialGroup, Mentor, Ohio). The sieves were based on US standard sieve sizesof 10, 18, 35, and 60 for 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm,respectively. The final filtrate passing through the sieves (less than0.25 mm fraction) is collected in 50 ml conical centrifuge tubes andcentrifuged at 4,000 rpm (about 4,000×g) for 10 minutes at 4° C. Thesupernatant is discarded and the pellet is suspended in one half theoriginal volume of PBS (e.g. 125 ml) containing 10% glycerol. Thesamples are used immediately, or stored frozen at −80° C. and thawed onice before transplantation.

Example 4

This example reports clinical experience with 43 consecutive patientsthat were treated for recurrent CDI C. difficile infection (CDI). Duringthis time donor identification and screening was simplified by movingfrom patient-identified individual donors to standard volunteer donors.Material preparation shifted from the endoscopy suite to a standardizedprocess in the laboratory, and ultimately to banking frozen processedfecal material that is ready to use when needed.

Standardization of material preparation significantly simplified thepractical aspects of treatment without loss of apparent efficacy inclearing recurrent CDI. Approximately 30% of the patients had underlyinginflammatory bowel disease, and treat went was equally effective in thisgroup. Several key steps in standardization of donor materialpreparation significantly simplified the clinical practice of treatmentof recurrent CDI in patients failing antibiotic therapy. This is alsoreported in Hamilton et al., Am. J. Gastroenterol., 2012,doi:10.1038/ajg.2011.482.

Methods

Patients

This report includes the first 43 patients who received fecal microbiotatransplantation (FMT) for recurrent CDI. All patients were identified bydirect referral from clinicians at infectious disease andgastroenterology practices in the Minneapolis and St. Paul metropolitanarea. Inclusion criteria for FMT included a history of symptomatic,toxin-positive, infection by C. difficile and at least two documentedsubsequent recurrences despite use of standard antibiotic therapy. Atleast one failed antibiotic regimen had to include a minimum of a 6 weekcourse of tapered or pulsed vancomycin dosage, or at least a one monthvancomycin course followed by a minimum two week rifaximin “chaser.” Theonly exclusion criteria in the protocol were age <18 and medicalfragility from non-C. difficile problems resulting in life expectancy of<1 year. In the latter situation we advised patients that the besttherapeutic option was an indefinite course of vancomycin. All patientsgave informed consent for FMT via colonoscopy, recognizing relativelylimited experience with this treatment approach and the intrinsicunknowns associated with its use. The Institutional Review Board at theUniversity of Minnesota approved prospective collection of clinicaloutcome data, while recognizing this experience does not constitute aclinical trial, and as such was not designed to test the efficacy of FMTin comparison with any other therapeutic options.

Donor Identification and Screening

At the start of the program patients were asked to self-identifypotential donors. These included mothers (n=2), daughters (n=1), sons(n=3), wives (n=1), husbands (n=1), and friends (n=2). Prior torecruitment, the donors were required to submit available medicalrecords and have a separate medical history interview away from therecipient patient. The history included: assessment of infectious risk,including identification of known risk factors for HIV and Hepatitis,current communicable diseases, and recent travel to areas of the worldwith a higher prevalence of diarrheal illnesses. Additional absolutedonor exclusion criteria included gastrointestinal co-morbidities andthe use of antibiotics within preceding three months. Since gutmicrobiota are likely involved in various aspects of energy metabolismand the functioning of the immune system, the presence of features ofmetabolic syndrome, autoimmunity, or allergic diseases were treated asrelative exclusion criteria. Donors provided separate informed consentto participate in the protocol, which included risks associated withlaboratory screening. The donors underwent serologic testing for HIV andHepatitis B and C, and stool testing that included screening for routineenteric pathogens, C. difficile toxin B, and examination for ova andparasites, and Giardia and Cryptosporidium antigens.

Given varying logistic difficulties in recruiting individualpatient-identified donors, the lack of availability of donor materialswhen needed, and no evidence to suggest a clear therapeutic advantage ofusing a related versus unrelated donor (e.g., son or daughter versusfriend or domestic partner), volunteer donors were recruited into theFMT program. The advantages of this change included removing the burdenof donor identification from the patient, improving the efficiency andcosts related to donor screening, a more consistent supply donor fecalmicrobiota, and the ability to impose extensive and stringent exclusioncriteria on donor selection (Table 2). Two unpaid volunteer donors wererecruited during this period, and one of them provided the majority ofdonated fecal material. Donor medical history was reviewed prior toevery donation and complete laboratory screening, as described above,was done every 6 months.

TABLE 2 Donor exclusion criteria. Donor Exclusion Criteria History andPhysical Examination Laboratory Screening Risk of Infectious 1. KnownHIV or Hepatitis B, C infection. 1. Ab for HIV 1 and 2. Agent 2. Knownexposure to HIV or viral 2. Viral Hepatitis A IgM. hepatitis at anytime. 3. Hepatitis B surface Ag and core Ab. 3. High risk behaviorsincluding sex for 4. HCV Ab. drugs or money, men who have sex with 5.RPR. men, more than one sexual partner in the 6. Stool cultures forenteric pathogens preceding 12 months, history of including Salmonella,Shigella, incarceration, any past use of Yersinia, Campylobacter, E.Colt intravenous drugs or intranasal cocaine. 0157:H7. 4. Tattoo or bodypiercing within 12 7. Ova and parasites examination. months. 8. Positivestool Giardia, 5. Travel to areas of the world with Cryptosporidium andHelicobacter increased risk of traveler's diarrhea. pylori antigens. 6.Current communicable disease, e.g., 9. Clostridium difficile toxin BPCR. upper respiratory tract viral infection. 10. Liver function testsincluding alkaline phosphatase, AST, ALT. Gastrointestinal 1. History ofirritable bowel syndrome, or comorbidities any of the associatedsymptoms, including frequent abdominal cramps, excessive gas, bloating,abdominal distension, fecal urgency, diarrhea or constipation. 2.History of inflammatory bowel disease such as Crohn's disease,ulcerative colitis, lymphocytic colitis. 3. Chronic diarrhea. 4. Chronicconstipation or use of laxatives. 5. History of gastrointestinalmalignancy or known colon polyposis. 6. History of any abdominalsurgery, e.g., gastric bypass, intestinal resection, appendectomy,cholecystectomy, etc. 7. Use of probiotics or any other over the counteraids for specific purposes of regulating digestion. Systemic Medical 1.Established metabolic syndrome or any 1. Serum triglycerides (>150mg/dL). Conditions early features suggestive of its 2. HDL cholesterol<40 mg/dL (males) emergence. Body mass index >26 kg/m2, and <50 mg/dL(females). waste:hip ratio >0.85 (male) and 3. High sensitivity CRP >2.4mg/L. >0.8 (female); BP >135 mmHg systolic 4. Fasting plasmaglucose >100 mg/dL. and >85 mmHg diastolic. 5. Liver function tests,including 2. Known systemic autoimmunity, e.g., alkaline phosphatase,AST, ALT. connective tissue disease, multiple 6. FANA. sclerosis, etc.3. Known atopic diseases including asthma or eczema. 4. Chronic painsyndromes including fibromyalgia, chronic fatigue syndrome 5. Ongoing(even if intermittent) use of any prescribed medications, includinginhalers or topical creams and ointments. 6. Neurologic,neurodevelopmental, and neurodegenerative disorders including autism,Parkinson's disease, etc. 7. Presence of a skin rash, wheezing onauscultation, lymphadenopathy, hepatomegaly or any stigmata of liverdisease, swollen or tender joints, muscle weakness, abnormalneurological examination. Additional factors 1. Antibiotics for anyindication within the known to affect the preceding 6 months.composition of intestinal microbiota

Donor Material Preparation

Individual patient-identified donors used in the early phase of theprogram came into the outpatient endoscopy center 1-2 h prior to thescheduled procedure.

The fecal material was collected in a toilet hat and processed in adedicated bathroom separate from the procedure room. Approximately 50 gmof fecal material was placed into a standard commercial blender (Oster,Sunbeam Corp, Rye, N.Y.) and homogenized in 250 mL of sterile,nonbacteriostatic nominal saline. The slurry was then passed throughstainless steel tea strainers to remove larger particles that couldinterfere with loading the syringes.

The material obtained from volunteer “universal” donors was transportedon ice into the laboratory, where it was processed within two hours ofcollection. The material was weighed and homogenized in a commercialblender in a dedicated biological cabinet. The slurry was then passedthrough 2.0 mm, 1.0 mm, 0.5 mm, and 0.25 mm stainless steel laboratorysieves (W. S. Tyler, Inc., Mentor, Ohio) to remove undigested food andsmaller particulate material. The resulting material passing through the0.25 mm sieve was centrifuged at 6,000×g for 15 min in a Sorvall SS-34rotor and resuspended to one half the original volume innonbacteriostatic natural saline. The resulting concentrated fecalbacteria suspension was administered to the patient immediately oramended with sterile pharmaceutical grade glycerol (Sigma, St. Louis,Mo.) to a final concentration of 10%, and stored frozen at −80° C. forone to eight weeks until used. Thawing was done over 2-4 hours in an icebath prior to the FMT procedure. The frozen preparation was diluted to250 ml with nonbacteriostatic normal saline prior to infusion in thedonor. This fecal material extract, whether fresh or frozen, was nearlyodorless and of reduced viscosity, color, and texture relative toearlier material prepared in the endoscopy center. Filtration of donormaterial allowed for effortless loading of large tip 60 mL syringeswithout risk of clogging. All containers, bottles, and sieves used inmaterial preparation were sterilized prior to use. Fecal material fromuniversal donors was treated in the same manner as that obtained frompatient-identified donors.

Transplantation Procedure

Patients were maintained on full dose of vancomycin (125 mg, 4 timesdaily, by mouth) until two days prior to the FMT procedure. The daybefore the procedure the patients were prepped using a split dosagepolyethylene glycol purge (GoLYTELY or MoviPrep), which is standard inour endoscopy unit, prior to colonoscopies to wash out residualantibiotic and fecal material. The patients underwent a full colonoscopyunder conscious sedation. Mucosal biopsies were taken to rule outlymphocytic colitis in absence of obvious inflammatory bowel disease.The majority of the prepared donor material (220-240 mL) wasadministered via the colonoscope's biopsy channel into the patient'sterminal ileum and cecum. In some cases, however, a small portion (50mL) was also instilled into colonic areas containing maximaldiverticulosis. Recovery procedure was identical to that routinely usedfor standard colonoscopy patients. All patients were instructed tocontact the endoscopist in case of symptom recurrence, were formallyfollowed in clinic 1-2 months after the procedure. Clearance of CDI wasdefined by resolution of diarrhea and negative stool testing for C.difficile at 2 months following FMT. All patients in this protocol alsoparticipated in a study examining fecal bacterial community structure,which involved collection of fecal specimens on days 3, 7, 14 and 1, 3,6, and 12 months after the procedure. The research staff collected thesespecimens from the patient's places of residence, providing additionalopportunities for symptom follow-up.

Statistical Analysis

Non-categorical data were compared using unpaired Student's t-test.Categorical data were compared using Fisher's exact test. GraphPad Prismsoftware was used to calculate two-tailed and two-sided p-values thatwere calculated with each test, respectively.

Results

Demographics

The group of patients with recurrent CDI described in this reportclearly had refractory disease as evidenced by the average number ofsequential relapses and duration of the condition (Table 3).Furthermore, many patients had multiple risk factors for highprobability of recurrence, such as history of severe CDI as evidenced byhospitalization, frequent use of non-C. difficile intercurrentantibiotics, and advanced age (Hu et al. Gastroenterology 2009;136:1206-14). All patients failed a long taper or pulsed regimen ofvancomycin, and 40% of patients also failed an additional long course ofvancomycin followed by a two-week rifaximin “chaser” regimen. One ofthese patients also failed a 4-week course of rifaximin. Severalpatients (3/43) took 2-4 week course of nita7oxanide, which also failedto clear the infection. Patients with inflammatory bowel disease werenot excluded from the protocol. Thirty five percent of our patients (14of 40) had underlying IBD, including Crohn's disease (6/14), ulcerativecolitis (4/14), and lymphocytic colitis (4/14). The patients with IBDwere generally younger (Table 4), but did not differ in the refractorynature of CDI or severity of presentation than older patients. However,the majority of patients without underlying 1BD had moderate to severediverticulosis.

TABLE 3 Demographics of patient population. The first 10 cases were doneusing patient-identified individual donors. After that, the protocolshifted to use of a standard donor. Fresh material was used in theearlier cases, and later practice shifted to use of frozen material.Duration Number (months) of Age of RCDI Relapses History of Donor (Mean± Female (Mean ± (Mean ± Hospitalization Interim Success Material SD)Gender SD) SD) for CDI Antibiotics PPI CRI IBD Diverticulosis RateIndividua 61 ± 22 70% 12.7 ± 7.3  6.2 ± 3.0 70% 60% 60% 30% 30% 50% 7/10 Donor (70%) (n = 10) Standard 55 ± 22 83% 13.1 ± 9.8  6.4 ± 3.375% 42% 33% 25% 50% 50% 11/12 Donor, (92%) Fresh Material (n = 12)Standard 59 ± 21 67% 10.1 ± 10.0 5.2 ± 3.0 38% 43% 43% 14% 24% 48% 19/21Donor, (90%) Frozen Material (n = 12) Total 59 ± 21 72% 12.2 ± 10.3 5.9± 3.3 56% 48% 47% 21% 33% 49% 37/43 Experience (86%) RCDI = Recurrent C.difficile Infection PPI = Proton Pump Inhibitor medication CRI = ChronicRenal Insufficiency or Failure IBD = Inflammatory Bowel Disease

TABLE 4 Comparison of patients without and with underlying IBD.Definition of IBD includes patients with Crohn's disease, ulcerativecolitis, and incidentally discovered lymphocytic colitis. Non-IBD (n =29) IBD (n = 14) p Value Age (Mean ± SEM) 64.7 ± 3.3 44.6 ± 5.8  p =0.0021 Female 69% 79% p = 0.43 (NS) Duration of RCDI 13.5 ± 2.1 8.3 ±3.3 0 = 0.09 (NS) (Mean # of months ± SD) Number of Relapses ±  6.2 ±3.0 4.4 ± 1.3 p = 0.04 SD Rate of Hospitalization 55% 57% P = 1.00 (NS)Interim Antibiotics 51% 36% p = 0.35 (NS) PPI 48% 43% p = 1.00 (NS)Renal Insufficiency 32% 14% p = 0.69 NS) Diverticulosis 69% 14% p =0.0028

Response to Treatment

The overall rate of infection clearance was 86% in response to a singleinfusion of donor fecal material, as evidenced by symptom resolution andnegative PCR testing for C. difficile toxin B after two months offollow-up (Table 3). Negative testing for C. difficile toxin B for twomonths was accepted as therapeutic success in patients with underlyingIBD, even in absence of complete symptom resolution. Three of tenpatients (30%) who received FMT using material from patient-identifiedindividual donors had a recurrence of CDI. Two standard donors wereemployed for the remaining 33 cases in this series, but the majority(30/33) were done using material prepared from a single donor. Three of33 patients who received FMT from a standard donor (fresh or frozen) hada recurrence of CDI. The difference in donor source, patient-identifiedversus standard, was not significant (p=0.1270). There was nosignificant difference in clearing the infection with fresh (11/12) orfrozen (19/21) donor material. All 6 patients who experienced recurrenceof CDI after FMT were offered a repeat procedure. Two of these patients,both >80 years of age, had multiple other active medical problems andpreferred to remain on indefinite treatment with vancomycin. Four otherpatients were treated with a second infusion, and all cleared theinfection bringing the overall success rate to 95% (41 of 43 patients).All second infusions were performed using the standard donor derivedmaterial. One of the recurrences of CDI occurred in a patient whoreceived his first infusion from the second standard donor. The samedonor source was used for his second FMT. Three of the four patients whoreceived a second FMT had underlying IRD; two patients had Crohn'sdisease and one had lymphocytic colitis. Finally, the fourth patient hada partial colon resection done for a stricture that developed followingher initial CDI episode. She has a colostomy draining her proximal colonand a long segment of residual distal colon. After recurrence of CDIwithin three weeks following her first FMT we thought it was likely thatengraftment in this case was complicated by difficulty in retaining thedonor material due to high flow of fecal contents and relatively smallsize of the infected colon. The second infusion in this case was donewith two doses of frozen standard donor material: one via the colostomyinto the colon and the other into the jejunum using upper pushenteroscopy. C. difficile testing of her fecal material was done weeklyin the first month and monthly thereafter. No C. difficile was foundover three months of follow-up.

No serious adverse events were noted following FMT in any of thepatients, with ether fresh or frozen materials. A minority of patients(approximately a third) noted some irregularity of bowel movements andexcessive flatulence during the first couple weeks following theprocedure, but these symptoms resolved by the time they were seen inclinic follow-up. Enhanced colitis activity in patients with underlyingIBD was not observed and there was improvement in overall colitisactivity in all patients with UC, although that is easily attributableto clearing the CDI. Interestingly, all diagnoses of lymphocytic colitiswere made for the first time from biopsies taken during thecolonoscopies performed at the time of FMT. These patients completelynormalized their bowel function and had no diarrhea after FMT withoutany additional medical therapy for lymphocytic colitis. Follow-upbiopsies were not performed in these patients when they becameasymptomatic.

Discussion

Recurrent infection is one of the most difficult clinical challenges inthe spectrum of C. difficile induced diarrheal disease. The risk ofrecurrence increases up to 65% after two or more episodes (McDonald etal. Emerg Infect Dis 2006; 12:409-15), and this risk is nearly certainin older patients who suffered severe CDI and suffered additionaldisruption of gut microbiota from intercurrent administration of non-C.difficile suppressing antibiotics (Hu et al. Gastroenterology 2009;136:1206-14). The inclusion criteria for patients in this case serieswere simple: at least three recurrences and failure of standardantibiotic treatments. Our patients averaged about six recurrences overan average course of one year. This population highlights known riskfactors for recurrence of CDI other than documented recurrence. Themajority had history of at least one hospitalization for severe CDI andalmost half took antibiotics after developing CDI for another non-C.difficile indication. Patients with inflammatory bowel disease dominatedthe younger age group. Virtually all patients were taking probiotics atpresentation and many have also tried toxin-binding resins. We did notsystematically collect information on all the various probioticspreparations taken by our patients, and many have tried multiple typesthrough the course of their recurrent infections. The most commonpreparations contained Saccharomyces boulardii and strains ofLactobacilli. All patients were recommended to discontinue takingprobiotics after FMT. In summary, by all available indicators thepatients in this case series had recalcitrant CDI that would not havehad a significant response rate to a placebo, and were unlikely torespond to another course of antibiotics or other available therapeuticoptions.

FMT has been used for decades as a last ditch method to cure recurrentCDI, and there has been growing uncontrolled evidence supporting itsefficacy. Here we report one of the largest single case series. The 95%overall success rate in this series is comparable to the cumulativeexperience in the literature (Bakken. Anaerobe 2009; 15:285-9, van Noodet al. Euro Surveill 2009; 14, Khoruts and Sadowsky. Mucosal Immunol2011; 4:4-7), and adds to the impetus for developing this therapeuticapproach to make it more widely available. The major issues tackled byour center were those of practicality. In the early phase of the programwe asked the patients to bring in prospective donors, which is the mostcommon approach in practice at this time. Our experience does notcontradict the efficacy of this approach. However, donor identificationand work-up increased expense of the procedure and introduced apotential delay period. Moreover, some patients who were alreadyexhausted by the illness had difficulty in finding suitable donors.While the ideal state of donor health may not be essential for elderlyrecipients with limited life expectancy, we felt compromise was not anoption for younger patients on any of the donor exclusion criteria. Gutmicrobiota constitute a human microbial organ with major functions inenergy metabolism and function of the immune system (Khoruts andSadowsky. Mucosal Immunol 2011; 4:4-7). Therefore, this transplantprocedure has potential implications for systemic physiology of therecipient. While donor health is not a guarantee to optimal compositionof gut microbiota, it is currently the only available indicator. For allthese reasons we decided to introduce the standard donor option to ourpatients. Interestingly, although many patients came into clinic withsome potential donor already identified, they all immediately preferredthe standard option of an anonymous screened donor upon learning aboutit.

The next challenge became advance preparation of the donor material.Little is known about viability of different constituents of fecalmicrobiota over time, and we did not wish to test this variable.However, since production of fresh material on demand is not alwayspractical, and does create delay and issues of sanitation andaesthetics, we introduced frozen donor material as another treatmentoption. The clinical efficacy of frozen preparation became quicklyevident and it has now become part of the standard protocol in ourprogram.

FMT is typically considered a last choice, desperate therapy option bymost clinicians, and to a great extent that is due to multiple aestheticand practical barriers that stand in the way of its administration.Increased prevalence, morbidity, and mortality of CDI has now reachedepidemic proportions and a significant fraction of these patients cannotclear the infection with standard therapies. These patients may benefitfrom FMT, but it is likely that the procedure is not available to them.Our FMT protocol has now progressed to the point where most obviousaesthetic and practical challenges have been overcome. This alsosignificantly reduces costs associated with screening of potentialdonors. While effort and organization is required for recruitment andscreening of suitable donors, as well as material preparation andbanking, execution of actual FMT has become a simple matter of loadingthe syringes with thawed, nearly odorless, material and a colonoscopy.

There are a number of limitations to this study. It was not a rigorousclinical trial designed to test efficacy of a particular FMT methodologyversus another, or some other form of therapy. Instead, it was anattempt to standardize FMT, as the procedure protocol evolved in thecourse of our clinical experience. Additional work is needed to readythis procedure for clinical trials and wider application. Nevertheless,our clinical outcomes provide very convincing evidence for efficacy ofthe frozen preparations. However, we cannot conclude from thisexperience alone that the fresh and frozen preparations are equivalent.The complexity of the donor material preparations, technical inabilityto culture most of the contained microbial constituents by classiclaboratory techniques, and our ignorance as to the identity of speciesthat are therapeutically most important precluded simple tests of donormaterial prior to FMT that could predict its efficacy. However, we arecurrently working to characterize the microbial composition of donormaterial and recipients' fecal samples collected over time by highthroughput 16S rRNA gene sequencing. Results of these experiments shouldprovide some means to compare different donor preparations. In addition,we are working to develop practical laboratory tests that will allow forfurther standardization of microbial composition of donor preparations.

While application of FMT for recurrent CDI has a long history, casereports suggest that it may also have a place in treatment of IBD andIBS (Bennet et al. Lancet 1989; 1:164, Borody et al. J ClinGastroenterol 2003; 37:42-7, Andrews and Borody. Med J Aust 1993;159:633-4). Given the potentially important role of gut microbiota inpathogenesis of the metabolic syndrome, FMT is already being explored ina clinical trial for this condition (Vrieze et al. Diabetologia 2010;53:606-13). Simplification and standardization of FMT-based therapeuticsis critical for its future development. Recent technological advanceshave also made it possible to gain insight into composition of gutmicrobiota and their activity. The study of microbiota in the context ofFMT should accelerate development of microbial therapeutics and yieldnew insights into microbial host interactions.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The invention claimed is:
 1. A method of decreasing the relative abundance of one or more members of the phylum Proteobacteria in a patient in need thereof and having a Clostridium difficile infection, the method comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a human fecal microbe preparation comprising a pharmaceutically acceptable carrier and a human fecal donor's intestinal microbiota comprising at least 6 different classes of bacteria selected from the group consisting of Actinobacteria, Bacteroidia, Bacilli, Clostridia, Erysipelotrichi, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Mollicutes, and Verrucomicrobiae, wherein said human fecal microbe preparation comprises particles of non-living material and particles of biological material, and said human fecal microbe preparation comprises no particle having a size of greater than 0.5 mm, wherein the relative abundance of one or more members of the phylum Proteobacteria is reduced by at least 10% following administration of said pharmaceutical composition.
 2. The method of claim 1, wherein said human fecal microbe preparation comprises an extract of feces from a human fecal donor.
 3. The method of claim 1, wherein said patient has a recurrent Clostridium difficile infection or is at risk of developing a recurrent Clostridium difficile infection.
 4. A method of decreasing the relative abundance of one or more members of the phylum Proteobacteria in a patient in need thereof, the method comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a fecal extract or preparation comprising a fecal donor's intestinal microbiota comprising at least 6 different classes of bacteria selected from the group consisting of Actinobacteria, Bacteroidia, Bacilli, Clostridia, Erysipelotrichi, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Mollicutes, and Verrucomicrobiae, wherein said fecal extract or preparation is capable of passing through a 0.5 mm sieve and further comprises a pharmaceutically acceptable carrier, wherein the relative abundance of one or more members of the phylum Proteobacteria is reduced by at least 10% following administration of said pharmaceutical composition.
 5. The method of claim 4, wherein said composition is contained within a capsule comprising at least one ingredient selected from the group consisting of a gelling agent, a plant polysaccharide or derivative thereof, a disintegrating agent, a lubricant, a glidant, a sweetening agent, a plasticizer, a surface treatment agent, a coloring agent, a preservative, and a flavoring agent.
 6. The method of claim 4, wherein said human fecal preparation further comprises an extract of feces from a human fecal donor.
 7. The method of claim 4, wherein said method also increases the fecal microbiota diversity in said patient.
 8. The method of claim 4, wherein said administering is orally administering or rectally administering.
 9. A method of decreasing the relative abundance of one or more members of the phylum Proteobacteria in a patient in need thereof, the method comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a human fecal microbe preparation comprising a pharmaceutically acceptable carrier and a human fecal extract comprising a human fecal donor's intestinal microbiota comprising at least 6 different classes of bacteria selected from the group consisting of Actinobacteria, Bacteroidia, Bacilli, Clostridia, Erysipelotrichi, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Mollicutes, and Verrucomicrobiae, wherein said human fecal extract comprises particles of non-living material and particles of biological material, and said human fecal extract comprises no particle having a size of greater than 0.5 mm, wherein the relative abundance of one or more members of the phylum Proteobacteria is reduced by at least 10% following administration of said pharmaceutical composition.
 10. The method of claim 9, wherein said method also increases the fecal microbiota diversity in said patient.
 11. The method of claim 9, wherein the relative abundance of said one or more members of the phylum Proteobacteria is decreased by at least 20% compared to said patient's colon before said administering.
 12. The method of claim 9, wherein the relative abundance of total members of the phylum Proteobacteria is decreased by at least 20% compared to said patient's colon before said administering.
 13. The method of claim 9, wherein the relative abundance is determined at a time selected from the group consisting of 3 days after said administering, 10 days after said administering, 15 days after said administering, and 25 days after said administering.
 14. The method of claim 9, wherein said administering is orally administering or rectally administering.
 15. The method of claim 14, wherein said rectally administering is selected from the group consisting of administering by a colonoscope, by enema, and by suppository.
 16. The method of claim 9, further comprising pretreating said patient with one or more antibiotics prior to said administering.
 17. The method of claim 16, wherein said one or more antibiotics are selected from the group consisting of Metronidazole, Rifaximin, Vancomycin, and Neomycin.
 18. The method of claim 9, wherein said human fecal preparation is derived from a human fecal donor unrelated to said patient.
 19. The method of claim 9, wherein said human fecal preparation comprises an extract of feces from a single human fecal donor.
 20. The method of claim 9, wherein said pharmaceutical composition is lyophilized.
 21. The method of claim 9, wherein said patient has a Clostridium difficile infection.
 22. The method of claim 9, wherein said patient has a recurrent Clostridium difficile infection.
 23. The method of claim 9, wherein said patient is at risk of developing a recurrent Clostridium difficile infection.
 24. The method of claim 9, wherein said pharmaceutical composition is contained within a capsule.
 25. The method of claim 9, wherein greater than fifty percent (50%) of bacterial species of said human fecal microbe preparation are members of the phylum Firmicutes.
 26. The method of claim 9, wherein said human fecal microbe preparation is dominated by the phylum Firmicutes.
 27. The method of claim 9, wherein the relative abundance of said one or more members of the phylum Proteobacteria is decreased in said patent's colon compared to said patient's colon before said administering.
 28. The method of claim 9, wherein the relative abundance of said one or more members of the phylum Proteobacteria is decreased in said patent's feces compared to said patient's feces before said administering. 